US4777132A - Assay for salicylate - Google Patents
Assay for salicylate Download PDFInfo
- Publication number
- US4777132A US4777132A US06/847,955 US84795586A US4777132A US 4777132 A US4777132 A US 4777132A US 84795586 A US84795586 A US 84795586A US 4777132 A US4777132 A US 4777132A
- Authority
- US
- United States
- Prior art keywords
- salicylate
- catechol
- sample
- hydroxylase
- concentration
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Expired - Lifetime
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
- C12N9/0004—Oxidoreductases (1.)
- C12N9/0071—Oxidoreductases (1.) acting on paired donors with incorporation of molecular oxygen (1.14)
- C12N9/0073—Oxidoreductases (1.) acting on paired donors with incorporation of molecular oxygen (1.14) with NADH or NADPH as one donor, and incorporation of one atom of oxygen 1.14.13
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/26—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving oxidoreductase
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Y—ENZYMES
- C12Y114/00—Oxidoreductases acting on paired donors, with incorporation or reduction of molecular oxygen (1.14)
- C12Y114/13—Oxidoreductases acting on paired donors, with incorporation or reduction of molecular oxygen (1.14) with NADH or NADPH as one donor, and incorporation of one atom of oxygen (1.14.13)
- C12Y114/13001—Salicylate 1-monooxygenase (1.14.13.1)
-
- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y10—TECHNICAL SUBJECTS COVERED BY FORMER USPC
- Y10S—TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y10S435/00—Chemistry: molecular biology and microbiology
- Y10S435/817—Enzyme or microbe electrode
Definitions
- the present invention is concerned with an assay for salicylate and with apparatus for performing the said assay.
- Aspirin acetyl salicylate
- Aspirin acetyl salicylate
- aspirin as a short term analgesic/antipyretic agent produces relatively low levels of salicylate in the serum (30-100 mg/l; 0.22-0.73 mM) and consequently monitoring of such levels is not normally necessary.
- This assay is based on the reaction of phenols with the Folin-Ciocalteau reagent in strong alkali solution to produce a blue colour which can be measured spectrophotometrically/colorimetrically (M. J. H. Smith & J. M. Talbot, Brit. J. Exp. Path., (1950), 31, 65).
- This method however requires the initial removal of protein from the serum samples and is not very specific for salicylate resulting in high "blank" values.
- Known methods for estimation of salicylate also include a first ultraviolet spectrophotometric method (L. Williams et al., J. Lab. Clin. Med., (1959), 53, 156), and a fluorophotometric method (A. Saltzman, J. Biol. Chem., (1948), 174, 399)
- a major disadvantage of such procedures is the need for expensive and bulky laboratory equipment.
- a method for the assay of salicylate or a derivative thereof which comprises the steps of:
- liquid sample is contacted with an electrode at a suitable potential which contacts the liquid sample for direct electrochemical measurement of the catechol.
- the hydroxylation and simultaneous decarboxylation of salicylate to yield catechol may be catalysed by any suitable enzyme of the type defined as Ec 1.14.13.1 and named as salicylate hydroxylase (otherwise known as salicylate 1-monooxygenase) by the International Union of Biochemistry (Enzyme Nomenclature, 1978, Academic Press, New York, (1979).
- the enzyme is usually a salicylate hydroxylase isolated from a bacterium, which is preferably a species of Pseudomonas, most especially Pseudomonas sp RPP (ATCC 29351) or Pseudomonas sp RWS (ATCC 29352).
- Such an enzyme material is preferably purified by ion-exchange chromatography, e.g. on an ion-exchange anion column.
- ion-exchange chromatography e.g. on an ion-exchange anion column.
- Fast protein ion exchange chromatography on a Polyanion SI column (Pharmacia) is of particular value.
- Any dissolved salicylate sample is susceptible of treatment in accordance with the invention.
- the sample comprises whole blood. It may, possibly, comprise plasma, serum, or any other like body fluid.
- the liquid sample may conveniently be contacted with an electrode having at its surface a layer comprising at least the said enzyme, usually in admixture with NADPH.
- a blood sample is applied onto the sensor. If the blood sample contains salicylate and the second substrate of the hydroxylase enzyme (NADH) is available for the enzyme (i.e. in the sample or on the electrode catalytic current is generated by the product (catechol) at the electrode surface. The potential is poised to oxidise the catechol and the current is measured.
- NADH hydroxylase enzyme
- Such electrodes themselves, especially when configured as a throw-away strip, and analytic equipment for salicylate, usable for whole blood samples, having such an electrode located or locatable therein also constitute aspects of the present invention.
- FIG. 1 is a postulated reaction scheme for the enzymic reaction
- FIG. 2 is a postulated reaction scheme for the reaction at the electrode surface
- FIG. 3 shows a cyclic voltammogram of catechol at 10 mM final concentration
- FIG. 4 shows a calibration curve for salicylate obtained by a series of steady state electrochemical measurements in the presence of increasing amounts of salicylate solution.
- FIG. 5 shows a cyclic voltammogram of NADH solution, salicylate hydroxylase solution and buffer solution, both in the absence and in the presence of salicylate,
- FIG. 6 shows the separation profile of Pseudomonad proteins by FPLC using ion exhange chromatography to obtain pure salicylate hydroxylase
- FIG. 7 shows a dry strip electrode response to salicylate.
- catechol (2) is converted into the orthoquinone (3) at the electrode surface and at a suitable oxidising potential.
- the removal of electrons from the catechol (2) results in the formation of ortho-quinone (3) or a derivative thereof, and may be employed both as a qualitative indicator of the presence of the catechol and hence the salicylate, and as a quantitative assay for the catechol and hence as an indirect measure of the concentration of salicylate at the electrode surface.
- a buffer solution was prepared from potassium di-hydrogen phosphate (1.77g; Analar from British Drug House (BDH) and di-potassium hydrogen phosphate (19.6 g; Analar from BDH), which were dissolved in distilled water, adjusted to pH 7.6 and made up to a final volume of 1 liter. Catechol (from Sigma Chemical Company) was dissolved in such a buffer solution and degassed under reduced pressure immediately prior to use.
- the electrodes were made of a range of different materials, especially gold and glassy carbon, most especially pyrolytic graphite.
- the electrodes were polished between runs using a slurry of 0.3 ⁇ m alumina (BDH) made up with water. The object of this polishing was to remove impurities and oxidation products from the surface of the electrode.
- the alumina was removed from the electrode surface by ultrasonication.
- Cyclic voltammograms were produced from a range of solutions by sweeping the potential difference from zero to +500 mV and back down to -100 mV vs. S.C.E.
- the potential applied was controlled by a potentiostat (from Jaytron Inst. A.S. Scientific. Abingdon) using a scan rate of 50 mv/s.
- the oxidation current produced was recorded on a Gould Series 60000 Chart Recorder in which the X-axis recorded the applied potential and the Y-axis recorded the current produced.
- a cyclic voltammogram of catechol (at 10 mM final concentration) is shown in FIG. 3.
- Salicylate sodium salt (GOLD LABEL and available in the marketplace from Aldrich) was dissolved in the phosphate buffer to give a final concentration of 0.1M.
- NADH disodium salt (Grade II; from Boehringer Mannheim) was dissolved in buffer solution to give a final concentration of 0.2M.
- Salicylate hydroxylase (from the Signal Chemical Company) was resuspended in distilled water to give a stock solution of 20 units/ml (based on the manufacturers information and unit definition).
- the electrodes used were identical to those described above with reference to Example 1.
- the cell contained 52 ⁇ l of NADH solution (0.2M, as above), 60 ⁇ l of salicylate hydroxylase solution and 428 ⁇ l of buffer solution.
- Cyclic voltammograms were recorded both in the absence and in the presence of the substrate (60 ⁇ l salicylate). In order to ensure that the reaction progressed, each sample was incubated at 37° C. for 2 minutes prior to initiating the scan.
- the stirred solutions comprised; 140 ⁇ l of NADH solution, 100 ⁇ l of salicylate hydroxylase solution and 760 ⁇ l of buffer solution.
- Steady state electrochemical measurements were made in the presence of increasing amounts of salicylate solution to produce a calibration curve for salicylate and is shown in FIG. 4.
- the current measured was obtained by poising the electrode at +250 mV vs S.C.E. 2 minutes after addition of the sample. This calibration curve can be used in conjuction with direct readings of unknown samples in order to determine the salicylate ion.
- a buffer solution was prepared from Trisma base (2.42g;Signal Chemical Company) dissolved in distilled water, adjusted to pH 7.5 and made up to a final volume of 1 liter. This buffer solution (buffer A) is used to apply the enzyme sample to the ion exchange column.
- a second buffer solution was prepared (buffer B). Buffer B was essentially the same as buffer A but also contains 150 mM sodium sulphate (BDH). This buffer is used to elute the enzyme from the anionic column.
- Salicylate hydroxylase from GDS Technology Inc. was resuspended in buffer solution A to give a stock solution of 50 units/ml (18 mg protein/ml) based on the manufacturers information and definition of activity and units.
- Protein purification was performed on a complete Pharmacia FPLC (Trade Mark) system.
- a Pharmacia Polyanion SI column HR5/5) was equilibrated with buffer A.
- the enzyme solution (16Oul) was applied to the column at a flow rate of lml min -1 .
- the sample was eluted from the column using a preprogrammed gradient (see FIG. 6).
- Fractions (lml) were collected in the FRAC-100 fraction collector (Pharmacia) and were assayed for enzyme activity as detailed in Example 2. Enzyme activity was present in fractions 20 and 21 and was associated with a protein peak. The profile of the chromatographic separation is shown in FIG. 6.
- the specific activity of the enzyme was in excess of 10 units/mg usually 14 to 15 units/mg.
- Sodium salicylate and NADH were obtained from the same sources as detailed in Example 2 and dissolved in 0.9% saline to give final concentrations of 20 mM. These two solutions were mixed in various proportions to give a range of salicylate concentrations in 10mM NADH.
- BES N'-Bis(2-hydroxyethyl-2-aminoethane sulphonic acids; 32.0 g from BDH), sodium azide (0.5 g;from BDH) and FAD disodium salt (85mg; from BDG) were dissolved in distilled water. adjusted to pH7 and made up to a final volume of 1 liter.
- the purified salicylate hydroxylase was ultraconcentrated using an Amicon ultrafiltration cell containing a 10,000 molecular weight cut-off filter and the buffer was concentrated to 520 units/ml.
- Dry strip electrodes were prepared according to British Patent Application No. 8515884.
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- Chemical & Material Sciences (AREA)
- Organic Chemistry (AREA)
- Life Sciences & Earth Sciences (AREA)
- Health & Medical Sciences (AREA)
- Engineering & Computer Science (AREA)
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- Bioinformatics & Cheminformatics (AREA)
- Genetics & Genomics (AREA)
- Biochemistry (AREA)
- General Health & Medical Sciences (AREA)
- General Engineering & Computer Science (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Molecular Biology (AREA)
- Microbiology (AREA)
- Biotechnology (AREA)
- Biomedical Technology (AREA)
- Physics & Mathematics (AREA)
- Analytical Chemistry (AREA)
- Biophysics (AREA)
- Medicinal Chemistry (AREA)
- Immunology (AREA)
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
- Steroid Compounds (AREA)
- Investigating Or Analyzing Materials By The Use Of Magnetic Means (AREA)
- Diaphragms For Electromechanical Transducers (AREA)
- Investigating Or Analyzing Materials By The Use Of Ultrasonic Waves (AREA)
- Organic Low-Molecular-Weight Compounds And Preparation Thereof (AREA)
- Investigating Or Analyzing Non-Biological Materials By The Use Of Chemical Means (AREA)
Abstract
Description
Claims (7)
Applications Claiming Priority (2)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
GB858508677A GB8508677D0 (en) | 1985-04-03 | 1985-04-03 | Assay for salicylate |
GB8508677 | 1985-04-03 |
Publications (1)
Publication Number | Publication Date |
---|---|
US4777132A true US4777132A (en) | 1988-10-11 |
Family
ID=10577124
Family Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
US06/847,955 Expired - Lifetime US4777132A (en) | 1985-04-03 | 1986-04-03 | Assay for salicylate |
Country Status (8)
Country | Link |
---|---|
US (1) | US4777132A (en) |
EP (1) | EP0202743B1 (en) |
JP (1) | JPH0650299B2 (en) |
AT (1) | ATE51415T1 (en) |
AU (1) | AU586235B2 (en) |
CA (1) | CA1253568A (en) |
DE (1) | DE3669888D1 (en) |
GB (1) | GB8508677D0 (en) |
Cited By (5)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US5320946A (en) * | 1990-07-05 | 1994-06-14 | Eastman Kodak Company | Method and element for assay of catechol and catechol generating substances |
US5362630A (en) * | 1981-07-28 | 1994-11-08 | Duke University | Isolation of pseudomonas salicylate hydroxlase and its use for the identification and quantitation of salicylate in body fluids |
US5460970A (en) * | 1993-05-18 | 1995-10-24 | Summa Health System | Separation of acetaldehyde-induced hemoglobin (Hb A1-AcH) |
US5736188A (en) * | 1994-08-08 | 1998-04-07 | Alcock; Susan | Printed fluid transport devices |
WO2000042421A1 (en) * | 1999-01-15 | 2000-07-20 | Competitive Technologies, Inc. | Method for screening for type b trichothecene mycotoxins |
Families Citing this family (1)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
DE19619056C2 (en) * | 1996-03-04 | 2002-01-17 | Frieder Scheller | Method and sensor for the enzymatic-electrochemical determination of substrates NAD · + · - and NAD (P) · + · -dependent dehydrogenases |
Citations (3)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US4416983A (en) * | 1980-12-11 | 1983-11-22 | Boehringer Mannheim Gmbh | Determination of NAD(P)H or salicylate |
CA1185155A (en) * | 1981-07-28 | 1985-04-09 | Kwan-Sa You | Isolation of pseudomonas salicylate hydroxylase and its use for the identification and quantitation of salicylate in body fluids |
EP0138530A2 (en) * | 1983-10-05 | 1985-04-24 | Secretary of State for Social Services in Her Britannic Majesty's Gov. of the U.K. of Great Britain and Northern Ireland | Method for the estimation of salicylates or reduced Pyridine nucleotides |
Family Cites Families (6)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
JPS5643358A (en) * | 1979-09-18 | 1981-04-22 | Tokuyama Soda Co Ltd | Pigment |
JPS57197458A (en) * | 1981-05-20 | 1982-12-03 | Yanagimoto Seisakusho:Kk | Catechol amine analysing apparatus |
JPS5926048A (en) * | 1982-08-03 | 1984-02-10 | Toshiba Corp | Sample inspection unit |
JPS5982082A (en) * | 1982-10-29 | 1984-05-11 | Matsushita Electric Works Ltd | Apparatus for determining glucose concentration |
AU564494B2 (en) * | 1983-05-05 | 1987-08-13 | Medisense Inc. | Enzyme cascade energy coupling assay |
AU564495B2 (en) * | 1983-05-05 | 1987-08-13 | Medisense Inc. | Nadp-nadph energy linked enzyme cascade assay |
-
1985
- 1985-04-03 GB GB858508677A patent/GB8508677D0/en active Pending
-
1986
- 1986-04-02 CA CA000505648A patent/CA1253568A/en not_active Expired
- 1986-04-03 AT AT86302476T patent/ATE51415T1/en not_active IP Right Cessation
- 1986-04-03 EP EP86302476A patent/EP0202743B1/en not_active Expired - Lifetime
- 1986-04-03 JP JP61075651A patent/JPH0650299B2/en not_active Expired - Fee Related
- 1986-04-03 US US06/847,955 patent/US4777132A/en not_active Expired - Lifetime
- 1986-04-03 AU AU55638/86A patent/AU586235B2/en not_active Ceased
- 1986-04-03 DE DE8686302476T patent/DE3669888D1/en not_active Expired - Fee Related
Patent Citations (3)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US4416983A (en) * | 1980-12-11 | 1983-11-22 | Boehringer Mannheim Gmbh | Determination of NAD(P)H or salicylate |
CA1185155A (en) * | 1981-07-28 | 1985-04-09 | Kwan-Sa You | Isolation of pseudomonas salicylate hydroxylase and its use for the identification and quantitation of salicylate in body fluids |
EP0138530A2 (en) * | 1983-10-05 | 1985-04-24 | Secretary of State for Social Services in Her Britannic Majesty's Gov. of the U.K. of Great Britain and Northern Ireland | Method for the estimation of salicylates or reduced Pyridine nucleotides |
Non-Patent Citations (16)
Title |
---|
Doskocil: Collection of Czechoslovak Chemical Communications 15, pp. 780 796, (1950). * |
Doskocil: Collection of Czechoslovak Chemical Communications 15, pp. 780-796, (1950). |
Fonong Analytica Chimica Acta 158, pp. 357 362 (1984). * |
Fonong Analytica Chimica Acta 158, pp. 357-362 (1984). |
Longenecker et al., Clin. Chem. 30(8):1369 1371 (1984). * |
Longenecker et al., Clin. Chem. 30(8):1369-1371 (1984). |
Power (1982) Anal. Chem. 54:1985 1987 (1985). * |
Power (1982) Anal. Chem. 54:1985-1987 (1985). |
Proudfoot Australian J. Chem. 36, pp. 885 894 (1983). * |
Proudfoot Australian J. Chem. 36, pp. 885-894 (1983). |
Rahni et al., Anal. Chim. Acta. 181:219 225 (1986). * |
Rahni et al., Anal. Chim. Acta. 181:219-225 (1986). |
You et al., Clin. Chem. 30(9):1549 1551 (1984). * |
You et al., Clin. Chem. 30(9):1549-1551 (1984). |
You, Clin. Chim. Acta. 149:283 285 (1985). * |
You, Clin. Chim. Acta. 149:283-285 (1985). |
Cited By (7)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US5362630A (en) * | 1981-07-28 | 1994-11-08 | Duke University | Isolation of pseudomonas salicylate hydroxlase and its use for the identification and quantitation of salicylate in body fluids |
US5580746A (en) * | 1981-07-28 | 1996-12-03 | You; Kwan-Sa | Isolation of pseudomonas salicylate hydroxylase and its use for the identification and quantitation of salicylate in body fluids |
US5320946A (en) * | 1990-07-05 | 1994-06-14 | Eastman Kodak Company | Method and element for assay of catechol and catechol generating substances |
US5506116A (en) * | 1990-07-05 | 1996-04-09 | Johnson & Johnson Clinical Diagnostics, Inc. | Method and element for assay of catechol and catechol generating substances |
US5460970A (en) * | 1993-05-18 | 1995-10-24 | Summa Health System | Separation of acetaldehyde-induced hemoglobin (Hb A1-AcH) |
US5736188A (en) * | 1994-08-08 | 1998-04-07 | Alcock; Susan | Printed fluid transport devices |
WO2000042421A1 (en) * | 1999-01-15 | 2000-07-20 | Competitive Technologies, Inc. | Method for screening for type b trichothecene mycotoxins |
Also Published As
Publication number | Publication date |
---|---|
GB8508677D0 (en) | 1985-05-09 |
EP0202743B1 (en) | 1990-03-28 |
EP0202743A1 (en) | 1986-11-26 |
CA1253568A (en) | 1989-05-02 |
AU586235B2 (en) | 1989-07-06 |
ATE51415T1 (en) | 1990-04-15 |
AU5563886A (en) | 1986-10-09 |
JPS6258157A (en) | 1987-03-13 |
DE3669888D1 (en) | 1990-05-03 |
JPH0650299B2 (en) | 1994-06-29 |
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